The invention relates to the introduction of a novel reagent for estimating biological toxins by in vitro test, as a replacement of currently used animal bioassay.
The direct testing of the toxins on animals poses ethical and moral questions. Therefore, there is great need for an alternative assay for biological toxins as a replacement for animal testing.
Biological toxins are grouped according to the source, such as: animal, plant, unicellular or one celled algae, and bacteria. Toxins are diverse and range from well-defined single macromolecules (tetanus, diphtheria, and botulinum toxins) to mixtures of complex molecules such as: snake or scorpion venoms or simple chemical entities (digitoxin, colchicine, tricyclic anti-depressants).
Mouse bioassay is the accepted and practiced method for assaying toxins, and is recommended by the Association of Official Analytical Chemists (AOAC). However, the mouse test for biological toxins is expensive and is disliked by animal activists and pressure is mounting worldwide to eliminate live animal bioassays. Germany, Switzerland and Australia have already banned the use of mammals for venom lethality tests.
The mouse bioassay suffers from other shortcomings as well. Toxins not lethal to mouse will go undetected by mouse bioassay. Also, mouse bioassay collectively detects the lethal effects of toxin, whether it is a single toxin or a mixture of several toxins.
Numerous investigators have developed immunological tests for assaying various toxins. Immunological tests are based on the principle of antigen-antibody reaction. Polyclonal antibodies are raised by immunizing animals: mouse, rabbit, rat, goat, etc. with a toxin.
Monoclonal antibodies are prepared in hybrid-oma cells. Spleen cells from an animal immunized with desired toxin are fused with corresponding species of myeloma cells. Both monoclonal and polyclonal antibodies produced are specific to the toxin (antigen used for immunization), and react with specific toxin.
The most frequently used in vitro immunological test is Enzyme Linked Immunosorbent Assay (ELISA). Biological toxins are assayed by several types of ELISA tests using specific antibodies to the desired toxin. ELISA tests can be carried out with numerous variations, although the most common format for detecting toxin from serum samples is the antigen-capturing method also known as double-sandwich method.
Li and Ownby reported the development of ELISA test for identification of venoms from snakes in the Agkistrodon genus. Several investigators have reported the development of ELISA for different toxins such as ricin and botulinum toxins; types A, B, and E in inoculated food samples. Morton and Tindall compared three tests; HPLC fluorescent method and two monoclonal antibody test kits for the determination of okadaic acid content of dinoflagellate cells and the results were not consistent. Since outbreaks of diuretic shellfish poisoning (DSP) may be caused by okadaic acid, methylokadaic acid, or a combination of these toxins, they concluded that both kinds of ELISA kits may underestimate total toxin effect in toxic shellfish.
All these methods suffer disadvantages, which have prevented their wide spread implementation particularly under the regulatory requirements. The mouse bioassay detects a wide range of known and presumably unknown toxins. Therefore, it is unlikely that it will be abandoned completely in favor of existing bioassays, until an alternative is found which is similarly responsive.
What is needed is a reagent that can be used in a common protocol for an in vitro assay test for biological toxins.
What is further needed is an in vitro test that can recognize all types of toxins, as a replacement of animal use.